Drugs targeting the orthosteric, primary binding site of G-protein coupled receptors are the most common therapeutics. Allosteric binding sites, elsewhere on the receptors, are less well-defined, and so less exploited clinically. We report the crystal structure of the prototypic beta-2 adrenergic receptor in complex with an orthosteric agonist and Compound-6FA, a positive allosteric modulator of this receptor. It binds on the receptor’s inner surface in a pocket created by intracellular loop 2 and transmembrane segments 3 and 4, stabilizing the loop in an alpha helical conformation required to engage the G-protein. Structural comparison explains the selectivity of the compound for beta-2 over the beta-1 adrenergic receptor. Diversity in location, mechanism, and selectivity of allosteric ligands provides potential to expand the range of receptor drugs.

Conventional drug discovery efforts at the β2-adrenoceptor (β2AR) have led to the development of ligands that bind almost exclusively to the receptor’s hormone-binding orthosteric site. However, targeting the largely unexplored and evolutionarily unique allosteric sites has potential for developing more specific drugs with fewer side effects than orthosteric ligands. Using our recently developed approach for screening G protein–coupled receptors (GPCRs) with DNA-encoded small-molecule libraries, we have discovered and characterized the first β2AR small-molecule positive allosteric modulators (PAMs)—compound (Cmpd)-6 [(R)-N-(4-amino-1-(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-(N-isopropyl-N-methylsulfamoyl)-2-((4-methoxyphenyl)thio)benzamide] and its analogs. We used purified human β2ARs, occupied by a high-affinity agonist, for the affinity-based screening of over 500 million distinct library compounds, which yielded Cmpd-6. It exhibits a low micro-molar affinity for the agonist-occupied β2AR and displays positive cooperativity with orthosteric agonists, thereby enhancing their binding to the receptor and ability to stabilize its active state. Cmpd-6 is cooperative with G protein and β-arrestin1 (a.k.a. arrestin2) to stabilize high-affinity, agonist-bound active states of the β2AR and potentiates downstream cAMP production and receptor recruitment of β-arrestin2 (a.k.a. arrestin3). Cmpd-6 is specific for the β2AR compared with the closely related β1AR. Structure–activity studies of select Cmpd-6 analogs defined the chemical groups that are critical for its biologic activity. We thus introduce the first small-molecule PAMs for the β2AR, which may serve as a lead molecule for the development of novel therapeutics. The approach described in this work establishes a broadly applicable proof-of-concept strategy for affinity-based discovery of small-molecule allosteric compounds targeting unique conformational states of GPCRs.